S176: FUNCTIONAL AND ANTIGEN-SPECIFIC CHARACTERIZATION OF IMMUNE CELLS AT THE SINGLE-CELL LEVEL REVEALS CONVERGENCE OF ADAPTIVE AND INNATE IMMUNITY IN IMMUNE APLASTIC ANEMIA
Authors: Jani Huuhtanen, Sofie Lundgren, Mikko Keränen, Xingim Feng, Alina Dulau-Florea, Bhavisha Patel, Yoshitaka Zaimoku, Cassandra Kerr, Emmi Jokinen, Markus Heinonen, Hanna Rajala, Sanna Siitonen, Freja Ebeling, Georgina Ryland, Lucy Fox, Piers Blombery, Eva Hellström-Lindberg, Jaroslaw P. Maciejewski, Neal S. Young, Harri Lähdesmäki, Satu Mustjoki
Journal: HemaSphere (2023)

Background: Immune aplastic anemia (AA) is a bone marrow failure (BMF) disorder driven by an autoimmune response against the hematopoietic stem and progenitor cells (HSPCs), yet the mechanisms of breaking self-tolerance and sustaining inflammation are incompletely understood. Aims: Characterize and functionally validate the immune cell phenotypes and T cell receptor (TCR) targets in AA and associate them with clinical variables. Methods: With scRNA+TCRαβ-seq, we profiled AA (n=13, N=17) from bone marrow (BM) and peripheral blood (PB) and compared these to healthy controls (HC) (n=35) and hematological malignancies (n=36). With TCRβ-seq, we profiled AA (n=144, N=248) from BM and PB and compared these to other BMFs (n=137), other autoimmune disorders (n=45), hematological malignancies (n=39), and HC (n=826, N=866). With multiplexed cytokine platform, we profiled AA (n=78, N=88) and HC (n=20). Findings were validated in co-cultures of HPSCs with immune cells with scRNA+TCRαβ-seq readout (AA n=7, HC n=4; N=57). The presence of clonal hematopoiesis, somatic loss of HLA alleles, and somatic mutations in the T cells were assessed in a subset of patients. Results: Compared to HC, the most statistically significant finding in the AA BM was the expansion of NK cell cluster. The increased proportion of NK cells was validated in a larger flow cytometry cohort (AA n=49, HC n=26) and was associated with more severe disease. NK cells had mostly terminally differentiated or adaptive NK cell phenotype with upregulation of cytotoxic markers and lineage-defining KLRC2/NKG2C and LAG3. Patients with the highest NK cell infiltration had also distinct cytokine profiles, with upregulated IL2/4/13/20 and CCL28. Compared to HC, patients with AA had also significantly more cytotoxic CD8+ T cells. The most expanded CD8+ T cell populations were terminally differentiated, highly cytotoxic CD8+ TEMRA cells, with upregulation of exhaustion markers (LAG3, TIGIT), but with retained stimulation capacity in vitro. Interestingly, these clonally expanded T cells also upregulated NK cell receptors, such as FCGR3A/CD16 and KLRC1/2/3. The expansion of these NK-like CD8+ TEMRA cells was validated in an additional scRNA-seq cohort (AA n=8, HC n=2) and was more pronounced in severe AA than in non-severe AA. The TCR-seq analysis showed oligoclonal expansions in patients with AA. With a sophisticated in-house TCR-seq analysis pipeline, we found 200 TCR-motifs that were associated with AA, and not found in HC, MDS, or other associated disorders or viral infections. These motifs were successfully validated in two large AA and HC TCR-seq cohorts and were associated with clinical variables such as loss of HLA class I alleles. T cells with AA-associated TCR motifs had NK-like CD8+ TEMRA phenotype, implying NK receptor expression in T cells could be a result of chronic antigen stimulation. Finally, the function of NK cells, NK-like CD8+ TEMRA cells, and clones with AA-associated TCR-motifs was assessed with autologous immune cell and HPSC co-cultures with scRNA+TCRαβ-seq readout and results will be presented at the conference. Summary/Conclusion: Our results show convergent shift of both innate and adaptive immune cells, where NK cells gain T cell-like memory properties and T cells gain NK-like TCR independent killing abilities to maintain autoinflammation in AA. Keywords: Autoimmunity, Aplastic anemia, T cell, Natural killer